Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genome that includes a large number of linear and circular plasmids. Abundant evidence indicates that some plasmid-encoded genes are critical for adaptation in the infectious cycle. A major focus of this project is to determine how and why the spirochete maintains such a unique genomic structure, and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. This is addressed in our laboratory primarily through a molecular genetic approach.[unreadable] [unreadable] The genome of B. burgdorferi comprises a single linear chromosome and approximately 20 linear and circular plasmids. Although some plasmids are required by B. burgdorferi for growth in its natural hosts, most plasmids are dispensable for growth in culture medium. However, one circular plasmid, cp26, is present in all natural B. burgdorferi isolates and has never been lost during propagation of the spirochete in the laboratory. We previously demonstrated that cp26 encodes essential functions required for growth under all conditions and hence cannot be lost or actively displaced from viable organisms. These essential functions provided by cp26 include, but are not limited to, the telomere resolvase required for replication of the chromosome. The identity of the other essential gene(s) on cp26 was not previously known. [unreadable] [unreadable] In FY2008, we used two different genetic techniques to identify those genes encoded on cp26 that are crucial for B. burgdorferi growth: targeted gene inactivation and plasmid displacement (Jewett et. al, Mol. Microbiol. 66:975-90, 2007). We attempted to knock out all cp26 genes to determine which functions encoded by this plasmid, in addition to telomere resolution, are required for B. burgdorferi growth in vitro. We then undertook to selectively displace cp26, using an incompatible plasmid that carried, along with resT, those genes implicated as important for bacterial survival. Through this systematic approach we have identified two genes on cp26 that together provide an essential function that is required for bacterial growth. These genes, designated bbb26 and bbb27, have no homologs in other organisms. We performed computational and experimental analyses to gain some insight into the putative essential role of bbb26 and bbb27. These studies indicated that both genes encode membrane-associated, periplasmic proteins that are minor components of the cell. Both proteins share some amino acid similarity to peptide-cleaving enzymes, but we have not detected any protease activity associated with them. In summary, we have determined that resT, bbb26 and bbb27 constitute the full complement of essential genes carried by cp26 that ensure its ubiquity in B. burgdorferi. Additional studies are required to identify the contribution of bbb26 and bbb27 to basic spirochete physiology.[unreadable] [unreadable] While cp26 carries genes important for bacterial growth, as described above, we have also previously shown that another gene on cp26, ospC, encodes a virulence factor that is absolutely required by B. burgdorferi for the initial colonization of the mammalian host. The linkage on cp26 of ospC, which is only required at one critical point during mammalian infection, with constitutively required functions encoded by resT, bbb26 and bbb27, ensures that ospC will not be lost during spirochete growth in environments in which it does not provide a selective advantage, such as during persistent mouse infection and within the tick vector. Indeed, we found that when the ospC gene is carried by a non-essential plasmid, it was lost by spirochetes in infected mice subsequent to the development of the acquired immune response of the host, thereby aborting the infectious cycle. In the current study described in this FY2008 report, we have identified the genes on cp26 that ensure stable retention of this essential plasmid by B. burgdorferi under the varying conditions encountered throughout the natural infectious cycle. We conclude that the genetic linkage of critical physiological and virulence functions on cp26 is pertinent to the evolution of this plasmid as an essential element of the B. burgdorferi genome.[unreadable] [unreadable] B. burgdorferi synthesizes a number of outer surface lipoproteins that are differentially regulated in the tick vector and vertebrate hosts. Among these is OspD, a protein that is highly induced in vitro by conditions that mimic the tick environment. Although OspD was first identified as a major surface protein of B. burgdorferi in 1992, a systematic examination of its expression and function in vivo had not previously been conducted. In FY2008, through genetic disruption of the ospD locus and analysis of RNA levels and protein synthesis patterns, we evaluated the requirement for this protein by B. burgdorferi throughout the mouse-tick transmission cycle (Stewart, Infect. Immun. 76:1970-8, 2008). Using genetically engineered strains deleted for ospD, we demonstrated that the protein encoded by this gene was not required for B. burgdorferi survival and infectivity in either the mammalian host or the tick vector. However, examination of both protein and transcript levels indicated that OspD expression by B. burgdorferi was limited to a brief period following tick detachment from the host after feeding, when spirochetes are actively replicating in the tick midgut. This discrete window of expression supports the finding that OspD is not required by the spirochete for mouse infectivity, as expression does not occur until after the tick has detached from the host. A reiterated sequence upstream of the ospD gene was previously hypothesized to constitute a putative regulatory sequence that could undergo recombination and alter gene expression. However, we observed no variation in this repeat region during B. burgdorferi infection of ticks, despite the dramatic changes in ospD gene expression that occur during this time and variation in the OspD phenotype of individual spirochetes.[unreadable] [unreadable] These results raise the question of why evolutionary pressures have not eliminated a gene such as ospD, which is likely to be energetically costly to the bacterium to regulate and express at a high level, yet appears to be nonessential to its lifestyle. B. burgdorferi has evolved a tight regulatory system for ospD that differs from other known outer surface proteins, which suggests a positive pressure for retention and dissemination. The timing of OspD synthesis correlates with the period of the highest cell densities observed for B. burgdorferi in vivo, which suggests a beneficial contribution of OspD to the spirochete during bacterial replication within the tick midgut. A possible function for OspD could relate to intercellular signaling or nutrient scavenging, which might provide an advantage in a natural tick infection where other bacteria are present. We conclude that the potential benefit provided by OspD outweighs the advantage of eliminating this nonessential locus and likely explains why it is widespread throughout Borrelia strains that cause Lyme disease.